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A comparative proteomic study identified LRPPRC and MCM7 as putative actors in imatinib mesylate cross-resistance in Lucena cell line.

Corrêa S, Pizzatti L, Du Rocher B, Mencalha A, Pinto D, Abdelhay E - Proteome Sci (2012)

Bottom Line: This resistance has been associated with the emergence of multidrug resistance (MDR) phenotype, as a BCR-ABL independent mechanism.The use of 2-DE coupled with a MS approach resulted in the identification of 36 differentially expressed proteins.In addition, our proteomic approach identified candidate actors involved in resistance, which could lead to additional information on BCR-ABL-independent molecular mechanisms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratório Célula-Tronco, Divisão de Laboratórios do CEMO, Instituto Nacional de Câncer, Rio de Janeiro, Brazil. scorrea@biof.ufrj.br.

ABSTRACT

Background: Although chronic myeloid leukemia (CML) treatment has improved since the introduction of imatinib mesylate (IM), cases of resistance have been reported. This resistance has been associated with the emergence of multidrug resistance (MDR) phenotype, as a BCR-ABL independent mechanism. The classic pathway studied in MDR promotion is ATP-binding cassette (ABC) family transporters expression, but other mechanisms that drive drug resistance are largely unknown. To better understand IM therapy relapse due to the rise of MDR, we compared the proteomic profiles of K562 and Lucena (K562/VCR) cells.

Results: The use of 2-DE coupled with a MS approach resulted in the identification of 36 differentially expressed proteins. Differential mRNA levels of leucine-rich PPR motif-containing (LRPPRC) protein, minichromosome maintenance complex component 7 (MCM7) and ATP-binding cassette sub-family B (MDR/TAP) member 1 (ABCB1) were capable of defining samples from CML patients as responsive or resistant to therapy.

Conclusions: Through the data presented in this work, we show the relevance of MDR to IM therapy. In addition, our proteomic approach identified candidate actors involved in resistance, which could lead to additional information on BCR-ABL-independent molecular mechanisms.

No MeSH data available.


Related in: MedlinePlus

Lucena cells cross-resistance to IM is due Pgp efflux. Apoptotic cells (A), cell cycle (B) and Rho 123 (C-D) were measured by flow cytometry after 3 different treatments conditions: 1 μM IM, 50 μM VP and co-treatment with 1 μM IM and 50 μM VP. (D) Representative histograms of Rho 123 extrusion under conditions described above. (1): K562 ctrl and IM treatment; (2): Lucena ctrl and (3) Lucena under: IM, VP and IM + VP treatments. K562 cells were used as positive control for Rho 123 retention, and K562 treated with 1 μM IM was used as positive control for apoptosis induction and cell cycle arrest. Values represent the means of three independent determinations ± s.d. (*p < 0.05; **p < 0.01). AF = auto fluorescence; K5 = K562; LU = Lucena.
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Figure 3: Lucena cells cross-resistance to IM is due Pgp efflux. Apoptotic cells (A), cell cycle (B) and Rho 123 (C-D) were measured by flow cytometry after 3 different treatments conditions: 1 μM IM, 50 μM VP and co-treatment with 1 μM IM and 50 μM VP. (D) Representative histograms of Rho 123 extrusion under conditions described above. (1): K562 ctrl and IM treatment; (2): Lucena ctrl and (3) Lucena under: IM, VP and IM + VP treatments. K562 cells were used as positive control for Rho 123 retention, and K562 treated with 1 μM IM was used as positive control for apoptosis induction and cell cycle arrest. Values represent the means of three independent determinations ± s.d. (*p < 0.05; **p < 0.01). AF = auto fluorescence; K5 = K562; LU = Lucena.

Mentions: Lucena cells were established from K562 cells as a multidrug resistance lineage by vincristine (VCR) selection and their pattern of resistance includes a range of chemotherapy drugs [19]. Real time quantitative PCR (RT-qPCR) analysis of K562 and Lucena cell lines showed an 800.0-fold increase of ABCB1 mRNA levels by Lucena cells compared to K562 cells (Figure 1A). Over-expression of Pgp protein level was examined by flow cytometry and we observed a 45.0-fold increase expression of Pgp in Lucena cells compared to parental cells (Figure 1B-C). As demonstrated by Assef and colleagues, K562 cells managed to exhibit a MDR phenotype through VCR treatment also presented cross-resistance to IM [20]. Because the development of MDR cell lines is not well described in the literature (i.e., different concentrations of chemotherapy drugs are used) and VCR maintenance concentrations differ among these cell lines, we investigated IM effectiveness in Lucena cells. We treated both cell lines with different concentrations of the drug for 24 h and assayed cell viability. A comparative analysis of the viability of K562 and Lucena cells after IM treatment with 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μM, 5 μM and 10 μM doses showed that Lucena cells were more resistant to IM than K562 cells (inhibitory concentration - IC50 5 μM and 1 μM, respectively; Figure 2A). To prove that the different cell viabilities were due to differential apoptotic activation between the cellular lineages, we conducted Annexin V assays with both cell lines under IM treatment with 1 μM (K562 IC50 dose). The results (Figure 2B) revealed that an IM dose of 1 μM activates apoptosis in approximately 20% of K562 cells but in only approximately 5% of Lucena cells. This result indicates that a 1 μM dose of IM for 24 h is insufficient to induce apoptosis in Lucena cells (Figure 2C) as it does in K562 cells. Moreover, cell cycle assay showed that IM treatment induced arrest in G0/G1 in both cell lines. However, this effect was more pronounced in K562 cells compared to Lucena cells (35.42% and 25.35%, respectively). Furthermore, we investigated the mRNA levels of BCR-ABL and the most related drug transporters involved in cancer with the goal of identifying the cause of Lucena cells cross-resistance to IM. In addition to possessing more ABCB1/Pgp than K562 cells (Figure 1), Lucena cells also had more BCR-ABL and OCT1 mRNA (Figure 2D), but did not presented significant difference in ABCG2 mRNA levels, which indicates that the failure of IM to induce apoptosis in Lucena cells may be due to Pgp drug efflux and/or by BCR-ABL up-regulation. In other to verify that cross-resistance to IM by Lucena cells might be through drug efflux, we performed apoptosis, cell cycle and Pgp activity assays on Lucena cells treated in 3 different conditions: 1 μM IM, 50 μM Verapamil (VP - Pgp blocker) and co-treated with 1 μM IM and 50 μM VP (Figure 3). VP treatment was not toxic and did not induce apoptosis (Figure 3A) and cell cycle arrest (Figure 3B) compared to control (untreated cells). Co-treatment enhanced cell cycle arrest in 10%, compared to IM alone (Figure 3B), which was followed by a 3.0-fold increase (approximately) in apoptosis compared to IM alone (Figure 3A). Rhodamine 123 (Rho 123) retention confirmed that K562 cells did not present functional Pgp efflux pump, even after IM treatment (Figure 3C-D). This accumulation was not verified in Lucena cells in both control (untreated cells) and IM treatment (Figure 3C-D), demonstrating that Pgp pump was functional in these cells. After VP treatment, irrespective of IM presence, Pgp was blocked, showing that Rho 123 was able to accumulate in these cells (Figure 3C-D).


A comparative proteomic study identified LRPPRC and MCM7 as putative actors in imatinib mesylate cross-resistance in Lucena cell line.

Corrêa S, Pizzatti L, Du Rocher B, Mencalha A, Pinto D, Abdelhay E - Proteome Sci (2012)

Lucena cells cross-resistance to IM is due Pgp efflux. Apoptotic cells (A), cell cycle (B) and Rho 123 (C-D) were measured by flow cytometry after 3 different treatments conditions: 1 μM IM, 50 μM VP and co-treatment with 1 μM IM and 50 μM VP. (D) Representative histograms of Rho 123 extrusion under conditions described above. (1): K562 ctrl and IM treatment; (2): Lucena ctrl and (3) Lucena under: IM, VP and IM + VP treatments. K562 cells were used as positive control for Rho 123 retention, and K562 treated with 1 μM IM was used as positive control for apoptosis induction and cell cycle arrest. Values represent the means of three independent determinations ± s.d. (*p < 0.05; **p < 0.01). AF = auto fluorescence; K5 = K562; LU = Lucena.
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Figure 3: Lucena cells cross-resistance to IM is due Pgp efflux. Apoptotic cells (A), cell cycle (B) and Rho 123 (C-D) were measured by flow cytometry after 3 different treatments conditions: 1 μM IM, 50 μM VP and co-treatment with 1 μM IM and 50 μM VP. (D) Representative histograms of Rho 123 extrusion under conditions described above. (1): K562 ctrl and IM treatment; (2): Lucena ctrl and (3) Lucena under: IM, VP and IM + VP treatments. K562 cells were used as positive control for Rho 123 retention, and K562 treated with 1 μM IM was used as positive control for apoptosis induction and cell cycle arrest. Values represent the means of three independent determinations ± s.d. (*p < 0.05; **p < 0.01). AF = auto fluorescence; K5 = K562; LU = Lucena.
Mentions: Lucena cells were established from K562 cells as a multidrug resistance lineage by vincristine (VCR) selection and their pattern of resistance includes a range of chemotherapy drugs [19]. Real time quantitative PCR (RT-qPCR) analysis of K562 and Lucena cell lines showed an 800.0-fold increase of ABCB1 mRNA levels by Lucena cells compared to K562 cells (Figure 1A). Over-expression of Pgp protein level was examined by flow cytometry and we observed a 45.0-fold increase expression of Pgp in Lucena cells compared to parental cells (Figure 1B-C). As demonstrated by Assef and colleagues, K562 cells managed to exhibit a MDR phenotype through VCR treatment also presented cross-resistance to IM [20]. Because the development of MDR cell lines is not well described in the literature (i.e., different concentrations of chemotherapy drugs are used) and VCR maintenance concentrations differ among these cell lines, we investigated IM effectiveness in Lucena cells. We treated both cell lines with different concentrations of the drug for 24 h and assayed cell viability. A comparative analysis of the viability of K562 and Lucena cells after IM treatment with 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μM, 5 μM and 10 μM doses showed that Lucena cells were more resistant to IM than K562 cells (inhibitory concentration - IC50 5 μM and 1 μM, respectively; Figure 2A). To prove that the different cell viabilities were due to differential apoptotic activation between the cellular lineages, we conducted Annexin V assays with both cell lines under IM treatment with 1 μM (K562 IC50 dose). The results (Figure 2B) revealed that an IM dose of 1 μM activates apoptosis in approximately 20% of K562 cells but in only approximately 5% of Lucena cells. This result indicates that a 1 μM dose of IM for 24 h is insufficient to induce apoptosis in Lucena cells (Figure 2C) as it does in K562 cells. Moreover, cell cycle assay showed that IM treatment induced arrest in G0/G1 in both cell lines. However, this effect was more pronounced in K562 cells compared to Lucena cells (35.42% and 25.35%, respectively). Furthermore, we investigated the mRNA levels of BCR-ABL and the most related drug transporters involved in cancer with the goal of identifying the cause of Lucena cells cross-resistance to IM. In addition to possessing more ABCB1/Pgp than K562 cells (Figure 1), Lucena cells also had more BCR-ABL and OCT1 mRNA (Figure 2D), but did not presented significant difference in ABCG2 mRNA levels, which indicates that the failure of IM to induce apoptosis in Lucena cells may be due to Pgp drug efflux and/or by BCR-ABL up-regulation. In other to verify that cross-resistance to IM by Lucena cells might be through drug efflux, we performed apoptosis, cell cycle and Pgp activity assays on Lucena cells treated in 3 different conditions: 1 μM IM, 50 μM Verapamil (VP - Pgp blocker) and co-treated with 1 μM IM and 50 μM VP (Figure 3). VP treatment was not toxic and did not induce apoptosis (Figure 3A) and cell cycle arrest (Figure 3B) compared to control (untreated cells). Co-treatment enhanced cell cycle arrest in 10%, compared to IM alone (Figure 3B), which was followed by a 3.0-fold increase (approximately) in apoptosis compared to IM alone (Figure 3A). Rhodamine 123 (Rho 123) retention confirmed that K562 cells did not present functional Pgp efflux pump, even after IM treatment (Figure 3C-D). This accumulation was not verified in Lucena cells in both control (untreated cells) and IM treatment (Figure 3C-D), demonstrating that Pgp pump was functional in these cells. After VP treatment, irrespective of IM presence, Pgp was blocked, showing that Rho 123 was able to accumulate in these cells (Figure 3C-D).

Bottom Line: This resistance has been associated with the emergence of multidrug resistance (MDR) phenotype, as a BCR-ABL independent mechanism.The use of 2-DE coupled with a MS approach resulted in the identification of 36 differentially expressed proteins.In addition, our proteomic approach identified candidate actors involved in resistance, which could lead to additional information on BCR-ABL-independent molecular mechanisms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratório Célula-Tronco, Divisão de Laboratórios do CEMO, Instituto Nacional de Câncer, Rio de Janeiro, Brazil. scorrea@biof.ufrj.br.

ABSTRACT

Background: Although chronic myeloid leukemia (CML) treatment has improved since the introduction of imatinib mesylate (IM), cases of resistance have been reported. This resistance has been associated with the emergence of multidrug resistance (MDR) phenotype, as a BCR-ABL independent mechanism. The classic pathway studied in MDR promotion is ATP-binding cassette (ABC) family transporters expression, but other mechanisms that drive drug resistance are largely unknown. To better understand IM therapy relapse due to the rise of MDR, we compared the proteomic profiles of K562 and Lucena (K562/VCR) cells.

Results: The use of 2-DE coupled with a MS approach resulted in the identification of 36 differentially expressed proteins. Differential mRNA levels of leucine-rich PPR motif-containing (LRPPRC) protein, minichromosome maintenance complex component 7 (MCM7) and ATP-binding cassette sub-family B (MDR/TAP) member 1 (ABCB1) were capable of defining samples from CML patients as responsive or resistant to therapy.

Conclusions: Through the data presented in this work, we show the relevance of MDR to IM therapy. In addition, our proteomic approach identified candidate actors involved in resistance, which could lead to additional information on BCR-ABL-independent molecular mechanisms.

No MeSH data available.


Related in: MedlinePlus